Method for unwinding high molecular weight whole-genome superhelix

Abstract

This invention relates to an unwinding macromolecule entire genome helical structure method, which belongs to the biology gene technique filed. This invention is to improve the technique of the nuclei acid microscopy spreading technique: óƒ improving the SLS in the spreading liquid by 5 times; ólowering the C chroma of the cell pigment to 50mg / mL; ó� mixing with proper hydro-cutting power. This invention can obtain the unwinding entire genome helical structure fully at one time, and display clearly combining with the special mutual protein (enzyme) molecule of the genome molecule, and can directly observe each of entire molecule information after the genome unwinding, such as molecular weight, in homogeneities and so on, and the obtained genome molecule back picture is clear, and its contrast is good. This technique can be used in various local making atlas constructing needed by the biology genome measuring, nucleic acid-protein mutual reaction research, genome structure research and other fields.

Description

technical field [0001] The invention discloses a method for unwinding the supercoiled structure of the whole genome with high molecular weight, which belongs to the technical field of biological genes, and in particular relates to a method for unwinding the supercoiled structure of the whole genome with high molecular weight. Background technique [0002] Biological genetic information is stored in genomic DNA molecules. Except for simple organisms, the molecular weight of DNA molecules in the genomes of most organisms is huge, which makes the expected whole genome sequencing work a huge project, labor-intensive and time-consuming. For example, the Human Genome Project (HGP) completed by scientists from six countries took more than 10 years. In the genome sequencing project, the task with the largest workload and the longest time-consuming is to draw the whole genome map (including genetic map, physical map, STS map, EST map, etc.) Half the time (the first 6 years of the H...

AI Technical Summary

Problems solved by technology

However, when these methods are used to spread high-molecular-weight genomes and achieve a completely unwound supercoiled structure, the results are not ideal, mainly in the following aspects: ① The genome is not completely unwound, and the obtained images have many supercoiled knots, and the number is difficult. distinguish

②The untwisted genome molecules often break, making it difficult to obtain complete genome molecules

③The conventional CytoC spreading method needs to add a higher concentration of CytoC to unfold the DNA molecule. The higher concentration of CytoC itself can cover the nucleic acid-protein complex, which makes it difficult to observe and study the nucleic acid-protein interaction, and this method is completely unwound. High-molecular-weight genomes remain difficult

④ BAC and ethidium bromide spreading methods can spread small molecular weight genomes to a certain extent and can be used to observe nucleic acid-protein complexes (due to the absence of CytoC), but knuckles often appear when spreading single-stranded genome molecules; When sampling, the sample on the copper grid is easily contaminated, the image contrast is low, the background is not clean and it is not easy to distinguish, the carbon film needs to be freshly made and needs to be activated, and the spread genome molecules have low affinity for uranyl acetate and acid phosphotungstic acid dyes

⑤ Zollinger et al. proposed the SLS-CytoC spreading method on the basis of summarizing previous work (see Zollinger M, Gaertin M, Mamet-Bratley MD. Anal Biochem, 1977, 82: 196-203), although this method can overcome BAC Compared with some shortcomings of the ethidium bromide spreading method, nucleic acid-protein complexes can also be observed, but the image background is still poor, and the nucleic acid-protein complexes are not clearly displayed (because the concentration of cytochrome C used has not yet effectively overcome the nucleic acid-protein complexes). The obscuring effect of protein complexes), and only suitable for the observation of small molecule genomes and their fragments

Images